ARTICLE
Received 29 Oct 2013 | Accepted 10 Apr 2014 | Published 27 May 2014
DOI: 10.1038/ncomms4867
OPEN
Conformational targeting of intracellular Ab
oligomers demonstrates their pathological
oligomerization inside the endoplasmic reticulum
Giovanni Meli1, Agnese Lecci1, Annalisa Manca1, Nina Krako1,2, Valentina Albertini3, Luisa Benussi3,
Roberta Ghidoni3 & Antonino Cattaneo1,2
Ab oligomers (AbOs) are crucially involved in Alzheimer’s Disease (AD). However, the lack of
selective approaches for targeting these polymorphic Ab assemblies represents a major
hurdle in understanding their biosynthesis, traffic and actions in living cells. Here, we
established a subcellularly localized conformational-selective interference (CSI) approach,
based on the expression of a recombinant antibody fragment against AbOs in the
endoplasmic reticulum (ER). By CSI, we can control extra- and intracellular pools of AbOs
produced in an AD-relevant cell model, without interfering with the maturation and
processing of the Ab precursor protein. The anti-AbOs intrabody selectively intercepts critical
AbO conformers in the ER, modulating their assembly and controlling their actions
in pathways of cellular homeostasis and synaptic signalling. Our results demonstrate that
intracellular Ab undergoes pathological oligomerization through critical conformations
formed inside the ER. This establishes intracellular AbOs as key targets for AD treatment and
presents CSI as a potential targeting strategy.
1 European Brain Research Institute (EBRI), Via del Fosso di Fiorano, 64, 00143 Roma, Italy. 2 Scuola Normale Superiore, Piazza dei Cavalieri, 7, 56126 Pisa,
Italy. 3 IRCCS ‘Centro S.Giovanni di Dio-Fatebenefratelli’, Via Pilastroni, 4, 25125 Brescia, Italy. Correspondence and requests for materials should be
addressed to A.C. (email: antonino.cattaneo@sns.it or a.cattaneo@ebri.it).
NATURE COMMUNICATIONS | 5:3867 | DOI: 10.1038/ncomms4867 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved.
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ARTICLE
T
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
he proteolytic processing, misfolding and self-aggregation
of several proteins are often associated with pathological
conditions and conformational diseases. However, the
identification, study and targeting of different conformations and
multimeric states of a given protein in the complex context of
subcellular compartments/microdomains of living cells remains a
big challenge.
A most relevant example of such a challenge is represented by
the amyloid-b (Ab) peptides, crucial players of Alzheimer’s
disease (AD) pathogenesis1–4. In living cells, differently than in a
test tube containing only synthetic peptides, the Ab species are
generated from the amyloid precursor protein (APP) by a
complex process of regulated intramembrane proteolysis (RIP)5,6,
and undergo a process of misfolding and aggregation, whose
mechanism and subcellular localization(s) are still debated7. In
particular, along the aggregation pathways, naturally occurring
Ab oligomers (AbOs) are still considered mysterious entities in
terms of molecular and structural composition and activity8, even
though they are recognized as the most neurotoxic proteinaceous
forms in AD8,9. As an additional level of complexity, APP–RIP
generates different proteolytic fragments and shares the secretases
(both a, b and g) with numerous different substrates5. Thus, the
cellular biochemistry of Ab is representative of two general
mechanisms operating in living cells and organisms, namely
RIP and amyloid formation (proteins enriched in cross b-sheet
and prone to self-aggregation) and represents not only a
pathologically relevant target, but also an ideal test case for the
development of new approaches for studying these processes
in cells.
Conformational-sensitive antibodies are important tools for
analysing amyloid assembly states and dynamics10. In particular,
recombinant antibody fragments can be exploited as intracellular
antibodies (intrabodies) for a subcellular-localized interference
to block or modulate the function of target molecules11,12. In
principle, if the intrabodies are intrinsically equipped with
conformational-sensitive binding properties, they could be
exploited for interference studies not currently feasible with
nucleic acid targeting methods (that is, RNA interference or gene
knockout), that can silence entire gene products (that is, APP
or RIP machinery protein components) but not peculiar posttranslational modification products (such as AbOs). Furthermore,
even though new chemical modulators and inhibitors for
APP–RIP or for Ab assembly are intensively studied, their
molecular selectivity and their precise subcellular delivery and
actions remain not easy to control. In the current state of the art,
while several conformation- and oligomeric-specific antibodies
targeting the Alzheimer’s AbOs have been developed8, they are
largely not exploitable for subcellular targeting and intracellular
functional studies in living cells.
We generated, by an in vivo intracellular selection in yeast
cells, a panel of conformation-sensitive antibody fragments
selectively recognizing AD-relevant AbO conformers13 ideal for
the expression (as genes) in mammalian cells, as intrabodies
targeted to different subcellular compartments. Here we
expressed the anti-AbO single-chain antibody fragment (scFv)
A13 (ref. 13) as an intrabody, with the aim of intercepting AbOs
at subcellular sites of their putative formation, and of attempting
their functional silencing. In this way, we established a
new experimental paradigm of subcellular-localized and
conformational-selective interference (CSI). The intrabodybased CSI besides providing a novel approach to selectively
control biologically-active AbO conformers in living cells, allows
a new dissection of cellular mechanisms of AbO generation,
trafficking and actions. Indeed, by exploiting CSI, we demonstrate
that intracellular Ab can oligomerize into pathological forms,
through critical conformations formed inside the endoplasmic
2
reticulum (ER). The anti-AbOs intrabody selectively intercepts
critical AbO conformers and controls their ‘toxic’ assembly in the
ER, without interfering with the complex processes of maturation
and processing of APP. Remarkably, the pool of targeted AbO
conformers are involved in the deregulation of two independent
pathways of cellular homeostasis and synaptic signalling. Our
overall results firmly establish the ER as the site of formation of
critical Ab conformers and validate ER-formed intracellular
AbOs as a key target for AD. For these reasons, the anti-AbOs
CSI can be exploitable for in vivo therapeutic applications as well
as to improve our understanding of the molecular and cellular
processes of AD pathogenesis, thereby uncovering new targets
for drugs development. Indeed, one of the earliest events
in AD pathogenesis seems to be the intraneuronal Ab
oligomerization14,15 and the synaptic targeting of AbOs3,16 and
current studies on the intracellular Ab generation and
oligomerization do not offer yet a common and conclusive
opinion, also for the lack of adequate experimental tools of study.
Results
The anti-AbO scFvA13 expressed as an intrabody in
mammalian cells. This study is based on scFvA13, a recombinant
antibody fragment generated by a direct intracellular selection of
functional intrabodies in yeast cells against the in vivo misfolded
antigen Ab13. The scFvA13 selectively recognizes conformational
Ab epitopes, as attested by the preferential binding in vitro to
synthetic oligomeric assemblies, such as the amyloid-derived
diffusible ligands (ADDLs17), but not to the monomeric or
fibrillar states13. Furthermore, the oligomeric-specific scFvA13purified protein was characterized for its neutralizing properties
against synthetic ADDLs13 or against endogenous Ab species18 as
well as for the recognition of pathological AbO deposits in the
brain of a mouse model of familial AD (fAD)19 and of other
neurodegeneration models20.
The conformation sensitivity of scFvA13 is accompanied by a
sequence specifity for Ab13, as also attested by dot blot (DB) assay
(Fig. 1a) and enzyme-linked immunosorbent assay (ELISA)
(Supplementary Fig. 1), which show the lack of immunoreactivity
with other proteins forming oligomers, such as a-Synuclein and
lysozyme, at variance with the generic anti-oligomer A11 (ref. 21)
(Supplementary Fig. 1).
Here, we tailored the scFvA13 for intrabody expression in
subcellular compartments of living cells12, to target critical
conformations of Ab oligomeric assemblies naturally occurring
inside cells and to intercept AbOs where they are formed in
the cell.
As a transmembrane protein, APP is cotranslationally inserted
in the membrane of the ER, where it traffics through the other
subcellular compartments of the secretory pathway; however, the
role of the ER in the APP–RIP and Ab generation, but especially
in Ab misfolding and oligomerization, remains to be deciphered
(Fig. 1b). We exploited the scFvA13 intrabody, to ask the
question as to where and when, during the APP maturation and
trafficking, is Ab formed and oligomerized, starting from the
early secretory compartment. To this aim, the scFvA13 was
targeted to the ER, through a C-terminal KDEL fusion allowing
its ER retention and its retrieval from the cis-Golgi, through the
ER-Golgi intermediate compartment12,22. In detail, the cDNA of
scFvA13-KDEL was stably expressed in the 7PA2 fAD cell
line23,24 (CHO cells overexpressing fAD human V717F APP
mutant, Supplementary Fig. 2), which represents a widely used
model of Ab oligomerization and the best characterized source of
natural, pathologically relevant and bioactive AbOs3,8,9,24–26.
Furthermore, the scFvA13-KDEL intrabody was expressed
in CHO transfectants expressing wild-type human APP referred
NATURE COMMUNICATIONS | 5:3867 | DOI: 10.1038/ncomms4867 | www.nature.com/naturecommunications
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ARTICLE
s
b
om
er
om
er
s
lig
ER
Ponceau mAb mAb
Aβ α-Syn
Lys
O
M
on
om
er
lig
O
M
on
om
er
s
a
s
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
α-Synuclein
Aβ1-42
Lysozyme
scFvA13
?
KDEL-R
Aβ
AβOs
scFvA13-KDEL
Secretory
APP
?
Aβ
Endosomes
Aβ
AβOs
AβOs
c
80.0
70.0
Intensity (uA)
60.0
50.0
Aβ1-17
Aβ1-18
Aβ1-19
Aβ11-40
Aβ11-42
Aβ1-33
Aβ1-34
Aβ1-37
Aβ1-38
Aβ1-39
Aβ1-40
Aβ1-42
…
d
40.0
10 kDa
30.0
20.0
3.5 kDa
CM CM
D4
A2
7W
7P
Aβ trimers
Aβ dimers
Aβ (4 kDa)
10.0
0.0
7WD4 CM
7PA2 CM
Figure 1 | The anti-AbOs scFvA13 exploited as intrabody in APP–RIP cell models. (a) Conformation sensitivity and sequence specificity of anti-AbOs
scFvA13. Monomers and oligomers of human a-synuclein, human Ab1-42 and lysozyme were assayed by DB with scFvA13 as primary antibody
(panel on the left). The scFvA13 is revealed through its C-terminal V5 tag. As a control, the same samples were respectively analysed through a
mAb anti a-synuclein (a-syn), the mAb anti-Ab 6E10 and by Ponceau S (panels on the right). The recognition of scFvA13 is selective and specific for
oligomers of human Ab1-42, but not for other proteins, as also shown by ELISA in Supplementary Fig. 1. (b) Cartoon illustrating APP–RIP (grey–light
blue-shaded box), Ab misfolding (green–red-shaded box), Ab oligomerization (box with a red frame) and trafficking in different (sub)cellular sites
(also presumptive, indicated by the question marks) as well as the localization of intrabody scFvA13-KDEL in the ER. a-, b- and g-secretase are represented
by yellow, orange and blue arrows, respectively and Ab misfolding by a change of a green fragment into a red hairpin. Some FAD mutations, such as the
hAPPV717F brought by the APP–RIP cell model 7PA2, alter the g-secretase cleavage determining pathological and unbalanced patterns of Ab peptides
(Supplementary Fig. 2). (c) Immunoproteomic (anti-Ab 6E10 þ 4G8 SELDI-TOF MS) profiles of Ab monomers and Ab truncated forms secreted by
7WD4 (CHO-hAPPwt) and 7PA2 (CHO-hAPPV717F) cells. The levels of Ab fragments and monomers are significantly higher in 7PA2 CM than in
7WD4 CM. Detailed ratios (7PA2/7WD4) for each Ab species are reported in Supplementary Table 1. Among these, the ratios for Ab1-40 and for Ab1-42
are 2.6 and 6.3, respectively. In addition, the fAD relevant Ab1-42:Ab1-40 ratio is B1:15 in 7WD4 and B1:6 in 7PA2. The C-terminal truncated forms
(ranging from Ab1-33 to Ab1-42) are the most abundant species detected in 7PA2 CM (Supplementary Table 1). The histogram shows the intensity
(microampere, uA) of single proteomic peaks. Mean values±s.e.m., n ¼ 4, Po0.001, Student’s t-test. (d) WB analysis (anti-Ab 6E10) of 7PA2 and
7WD4 CM (both 15 concentrated). Ab and AbOs (LDS/SDS-stable Ab dimers and trimers) are almost exclusively detected in 7PA2 CM.
to as 7WD4 (ref. 23) (Supplementary Fig. 2), which show much
lower rates of Ab production and oligomerization in comparison
with 7PA2 (Fig. 1c,d) and in human neuroblastoma SHSY5Y
cells, expressing only the endogenous APP, that show effective
RIP and negligible Ab oligomerization.
Measuring Ab and AbOs biochemical patterns in 7PA2 cells.
The detection of the pathologically relevant Ab and AbOs
biochemical patterns in 7PA2 cells is essential prerequisite to
study the effects of the subcellular targeting of scFvA13 intrabody.
An Ab immunoproteomic analysis, based on the use of anti-Ab
(monoclonal antibody (mAb) 6E10 and 4G8) as capturing
antibodies in SELDI-TOF mass spectrometry (MS)27, allows
directly revealing the complex profile of Ab monomers and
fragments secreted by cells in the conditioned medium (CM). In
this way, by comparing the 7PA2 and 7WD4 cells (respectively
expressing the V717F mutant or the wild-type human APP),
we measured in 7PA2 CM higher levels of secreted Ab
peptides (Fig. 1c) and, most importantly, a fAD-related higher
Ab1-42:Ab1-40 ratio (Fig. 1c, Supplementary Table 1). Of note,
we observed an overall unbalanced proportion of carboxy- and
amino-terminally truncated Ab peptides (Supplementary Table 1)
in 7PA2 CM, similar to Ab profiles in cerebrospinal fluids
from human fAD28 and representative of a fAD-linked APP
misprocessing (Supplementary Fig. 2).
The 6E10 þ 4G8 SELDI-TOF MS method reveals in 7PA2 CM,
the complex profile of Ab monomeric peptides and of Ab
truncated forms but does not detect immunoproteomic peaks
corresponding to AbOs. Thus, we investigated the patterns of
soluble Ab also by other approaches, such as western blot (WB)
or DB. The WB analysis of straight (non-concentrated) 7PA2 CM
shows that the Ab oligomeric bands are a small fraction (o20%)
of total soluble Ab species, which mainly run in the gels as
monomeric bands (Supplementary Fig. 3). Accordingly, the
biologically relevant LDS/SDS-stable Ab dimers and trimers were
previously recognized in 7PA2 CM as a small pool of total soluble
Ab species only upon immunoprecipitation enrichment23,24.
NATURE COMMUNICATIONS | 5:3867 | DOI: 10.1038/ncomms4867 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
a
scFvA13-KDEL
Calnexin
scFvA13-KDEL
58 K
b
1
2
3
4
5
6
7
8
9
10
11/12
Fraction no.
Calnexin
GM130
Rab3A
scFvA13-KDEL
c
L
L
ec DE
ec DE
-S 3-K
-S 3-K
3
3
A1 A1
A1 A1
Fv Fv
Fv Fv
sc sc
sc sc
d
K
13
3K
3K Y
-A
1
Y
5
5
-A
D4 D4
SY SY
A2 PA2
7
SH SH
7W 7W
7P
94 kDa (Grp94)
78 kDa (Grp78=BiP)
1
-A
55 kDa
scFvA13-KDEL (V5tag)
β-Actin
Lysates
Media
Figure 2 | The intrabody scFvA13-KDEL is targeted to the ER. (a) Confocal analysis of 7PA2 cells stably expressing the scFvA13-KDEL intrabody
(7PA2-A13K cells). The intrabody was detected by indirect immunofluorescence through its C-terminal V5 tag by using an anti-V5 as a primary
antibody (left panels). The middle panels show, from the top the ER marker calnexin and the Golgi marker protein 58K. The panels on the right show
the respective merged images (scFvA13-KDEL with calnexin or 58K) with a major co-localization of scFvA13-KDEL intrabody with calnexin, and a partial
co-localization of scFvA13-KDEL intrabody with 58K, indicating the preferential KDEL-mediated ER localization of the intrabody. Scale bar, 10 mm,
magnification 63. (b) WB analysis for calnexin (ER), GM130 (Golgi) and Rab3A (secretory vesicles) of a representative microsome fractionation of
7PA2-A13K cells. The scFvA13-KDEL intrabody is detected in the ER-enriched fractions. (c) WB analysis (blot with anti-V5) of lysates and of CM of
representative 7PA2 clones stably expressing the scFvA13-KDEL or the scFvA13 secretory intrabody. The intrabody KDEL expressing cells do not
secrete scFv protein in the culture media (no WB detection even in CM 15 concentrated). (d) WB analysis (by using anti-KDEL as primary antibody)
of KDEL proteins in 7WD4, 7PA2 and human neuroblastoma SHSY5Y, expressing or not the scFvA13-KDEL intrabody (steady-state levels). The
expression of the intrabody KDEL does not perturb the expression of other KDEL proteins, such as the ER chaperones (that is, Grp78 or BiP and Grp94),
showing it does not induce ER stress (BiP levels unchanged). WB of b-Actin and of scFv (by anti-V5) is also shown.
Here, in a new way, we were able to detect LDS/SDS-stable Ab
dimers and trimers by a direct WB analysis of 7PA2 CM
(Supplementary Fig. 3), with a major sensitivity if the CM is
previously subjected to concentration steps. We demonstrated
that the dimeric and trimeric bands are Ab-specific being
selectively abolished by cell pretreatment with a g-secretase
inhibitor (L-685,458)29 and being recognized by independent
anti-Ab antibodies (6E10 and 4G8) raised against different Ab
epitopes (Supplementary Figs 4,5).
Finally, we established a new AbO-specific DB assay, based on
the use of scFvA13, and of the other conformation-sensitive antiAbO scFvIm3 (ref. 13), as primary antibodies (Supplementary
Fig. 6). The scFv-based DB can selectively detect both
4
extracellular and intracellular Ab conformers (Supplementary
Figs 6,7). Of note, the DB approach is essential to analyse the
intracellular pools of AbOs, hardly detectable by WB both for
sensitivity limits and for the main cross-reactivity of the anti-Ab
6E10 and 4G8 with a large number of intracellular APP fragments
(Supplementary Fig. 7a). On the other hand, the anti-Ab WB is a
good method to detect Ab monomeric bands, both in lysates and
microsomes (Supplementary Fig. 7b), which are representative of
the largest pool of intracellular Ab species (both monomers and
LDS/SDS unstable oligomers).
Targeting the scFvA13 intrabody to the ER. The scFvA13
intrabody was targeted to the early secretory pathway exploiting
NATURE COMMUNICATIONS | 5:3867 | DOI: 10.1038/ncomms4867 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
a
120
7P
Aβ trimers
Aβ dimers
Aβ (4 kDa)
CM 2-A
A
7P
WB: 4G8
Aβ trimers
Aβ dimers
Aβ (4 kDa)
*
*
*
80
7PA2 CM
7PA2-A13K CM
60
40
20
WB: 6E10
0
Aβ
Aβ
Aβ
4 kDa dimers trimers
Aβ
Aβ
Aβ
4 kDa dimers trimers
WB: 4G8
b
*
3K
CM
CM -A1
A2 A2
7P 7P
DB: scFvA13
Aβ Oligomers
DB: scFvIm3
Levels of Aβ species
(% of 7PA2)
120
c
WB: 6E10
*
100
80
7PA2 CM
7PA2-A13K CM
60
40
20
0
Aβ oligomers
Aβ oligomers
DB: scFvA13
DB: scFvIm3
120
Aβ1-17
Aβ1-18
Aβ1-19
Aβ11-40
Aβ11-42
Aβ1-33
Aβ1-34
Aβ1-37
Aβ1-38
Aβ1-39
Aβ1-40
Aβ1-42
100
Intensity (uA)
*
100
Levels of Aβ species
(% of 7PA2)
K
13
A2
CM
80
60
40
20
0
7PA2 CM
7PA2-A13K CM
Figure 3 | Impact of scFvA13-KDEL in 7PA2 cells on the extracellular patterns of Ab species. (a) WB analysis of secreted AbOs in the CM of 7PA2 cells
and of 7PA2 stably expressing the scFvA13-KDEL intrabody (7PA2-A13K). LDS/SDS-stable Ab dimers and Ab trimers were detected by WB using two
different anti-Ab mAbs, mAb 4G8 (against a.a. 17–25 of Ab) and mAb 6E10 (against a.a. 3–9 of Ab), as primary antibodies (see also Supplementary Fig. 5).
Ab dimers and Ab trimers are significantly reduced in 7PA2-A13K, whereas no significant changes in the 4 kDa Ab bands (deriving both from monomers
and LDS/SDS unstable oligomers) are observed (see also Supplementary Figs. 3,5). The histogram shows the levels of Ab species in 7PA2-A13K CM in
comparison with 7PA2 CM (at 100) obtained from densitometric values of bands and normalized as discussed in the method section and in the
Supplementary Fig. 5. Mean values±s.e.m., n ¼ 4, *Po0.01, Student’s t-test. (b) DB analysis of AbOs in CM were done by using the conformationsensitive and sequence-specific scFvA13 or scFvIm3 anti-AbOs13 as primary antibodies (and detected through anti-His tag). The levels of AbOs are
significantly decreased in 7PA2-A13K CM, in comparison with 7PA2 CM, similarly to those of Ab dimers and Ab trimers (measured by WB). The histogram
shows the levels of AbOs in 7PA2-A13K CM in comparison with 7PA2 CM (at 100) obtained from densitometric values of dots and normalized as
discussed in the method section. Mean values±s.e.m., n ¼ 4, *Po0.01, Student’s t-test. (c) Immunoproteomic analysis of secreted Ab monomers and
fragments. The levels of N- and C-terminally truncated Ab monomers, measured by 6E10 þ 4G8 SELDI-TOF MS, are significantly increased in 7PA2-A13K
CM, in comparison with 7PA2 CM (all ratios in Supplementary Table 2). The most abundant Ab species are increased by 53.1±1.6% (Ab1-37), 44.2±1.1%
(Ab1-38), 48.6±0.9% (Ab1-39), 50±0.3% (Ab1-40), 59±3.3% (Ab1-42), respectively. The histogram shows the intensity (microampere, uA) of single
proteomic peaks. Mean values±s.e.m., n ¼ 4, Po0.001, Student’s t-test.
the ER retention/retrieval KDEL system. The subcellular
localization of scFvA13-KDEL was confirmed by indirect
immunofluorescence to be mainly in the ER and partially in
the cis-Golgi (Fig. 2a). The effective enrichment in the ER was
further demonstrated through the WB analysis of different
subcellular vesicles derived by microsome fractionation of
7PA2 scFvA13-KDEL transfectants (Fig. 2b). Consistent with
its retention in the ER, the scFvA13-KDEL was found not
to be secreted in the extracellular medium (Fig. 2c).
Of note, the scFvA13-KDEL intrabody does not perturb
the expression levels of endogenous ER KDEL-bearing proteins
or chaperones, and does not induce ER stress in 7PA2 and
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
3K
7P
Aβ
7P
*
*
100
WB: 4G8
WB: 6E10
β-Actin
DB: scFvA13
AβOs
*
120
A1
A2
DB: scFvIm3
Levels of Aβ species
(% of 7PA2)
A2
80
7PA2
7PA2-A13K
60
40
20
DB: pAb A11
0
Aβ
(4G8)
Aβ
(6E10)
AβOs
AβOs
AβOs
(scFvA13) (scFvIm3) (pAb A11)
Figure 4 | The intracellular Ab and AbOs in 7PA2-A13K cells. WB analysis of Ab (blot with 6E10 and 4G8) and DB analysis of AbOs (blot with the
anti-AbOs scFvA13 and scFvIm3 and with the generic anti-oligomers pAbA11) in 7PA2 and 7PA2-A13K cell lysates. In WB, the Ab monomeric bands
do not show significant changes (nor in total microsomes, see Supplementary Fig. 7b). The intracellular AbOs, measured by scFvA13 or scFvIm3 or
pAbA11 DB on cell lysates, show significantly decreased levels in 7PA2-A13K cells in comparison with 7PA2 cells. The histogram shows the Ab and
AbO levels in 7PA2-A13K lysates in comparison with 7PA2 cells (at 100) obtained from densitometric values of bands and dots and normalized
versus b-Actin and versus the amount of total proteins in lysates. Mean values±s.e.m., n ¼ 3, *Po0.01, Student’s t-test.
other cell lines (as measured by the unchanged levels of BiP)
(Fig. 2d).
The effect of scFvA13-KDEL on the extracellular pools of Ab
and AbOs. To better study the effects of scFvA13-KDEL on the
biochemistry of Ab, AbOs and APP, we first established 7PA2
cells stably expressing the intrabody scFvA13-KDEL, referred to
as 7PA2-A13K cells.
The comparative WB analysis of straight CM from 7PA2 and
7PA2-A13K cells does not reveal significant differences in the
levels of Ab monomeric bands, which are representative of the
largest amount of soluble Ab species (monomers and LDS/SDS
unstable oligomers) (Supplementary Fig. 3).
Instead, the amount of LDS/SDS-stable Ab dimers and trimers
secreted from 7PA2 cells (detected in concentrated CM) is
significantly reduced (B60% in 4G8-WB and B70% in 6E10WB) by the expression of the scFvA13-KDEL intrabody (Fig. 3a;
Supplementary Fig. 5). Similarly, the secreted oligomeric
conformers, measured by scFv-DB assay in CM, are significantly
reduced (70–80%) in 7PA2-A13K (Fig. 3b; Supplementary Fig. 6).
Of note, the similar ratio of AbOs decrease, measured by WB
(selective for Ab dimers and trimers with respect to the
monomeric Ab bands) and by DB, further attests the oligomeric
specificity of the scFv-based DB assay (in addition to the
experimental control of g-secretase inhibitor treatment,
Supplementary Fig. 6).
In contrast to the reduction of AbOs measured in the CM of
7PA2-A13K cells, the immunoproteomic analysis of 7PA2-A13K
CM (by 6E10 þ 4G8 SELDI-TOF MS) shows a significant
increase of Ab monomeric species, with respect to 7PA2 CM
(Fig. 3c). Relevantly, the expression of scFvA13-KDEL does not
alter the profile of g-secretase-generated Ab products (species of
37–42 amino-acid length) in 7PA2 cells, but leads, instead, to a
similar increase in the absolute amounts of all Ab species (Fig. 3c,
Supplementary Table 2).
Thus, the intracellular expression of scFvA13-KDEL determines a selective and significant reduction of extracellular AbOs
accompanied by an opposite increase of secreted Ab monomers
and truncated forms. While it is difficult to establish a precise
quantitative balance between these two independent Ab pools,
our results show that their variations are likely mutually
complementary.
Modulation of the intracellular Ab and AbOs pools by the
scFvA13-KDEL. We evaluated the effects of the scFvA13-KDEL
6
expression on the intracellular pools of Ab and AbOs by WB and
DB analyses. The anti-Ab WB analysis allows concluding that the
intracellular Ab monomeric bands are not significantly changed
in 7PA2-A13K lysates (Fig. 4), neither in total microsomes
(Supplementary Fig. 7b), while the levels of intracellular AbO
conformers (selectively detected by DB analysis in cell lysates) are
significantly decreased (Fig. 4, Supplementary Fig. 7b). Of note,
also the intracellular oligomers detected (in DB) by the
independent A11 polyclonal antibody21, known to recognize
the so-called ‘prefibrillar oligomers’ (larger than dimers and
trimers)30, are found to be decreased in 7PA2-A13K cells (Fig. 4).
Thus, the reduction of secreted AbOs determined by the
expression of scFvA13-KDEL occurs without a concomitant
intracellular accumulation of Ab and with a reduction of
intracellular AbO conformers, detected by scFvA13, scFvIm3
and A11 likely as distinct pools of AbOs.
Results can be explained by an interference of the scFvA13KDEL intrabody in the formation of stable AbOs in the ER,
highlighting the relevance of intracellular (as opposed to the
extracellular) Ab oligomerization process in the overall balance of
AbOs. We conclude that the ER-assembled AbOs are a consistent
source of those secreted extracellularly and of those found
intracellularly.
Of note, the expression of an Ab-unrelated intrabody (scFvR4KDEL used as negative control) in 7PA2 cells, has no effect on the
levels of extra- and intracellular Ab or AbOs (Supplementary
Fig. 8).
ScFvA13-KDEL intrabody does not affect the APP metabolism.
To further characterize the cellular mechanisms leading to the
observed changes in Ab/AbOs levels in the 7PA2-A13K cells
(summarized in the Supplementary Table 3), we investigated
whether the intrabody has a direct or indirect interference on the
expression, maturation and processing of APP, processes which
tightly influence the levels of secreted Ab (Fig. 5).
The steady-state levels of full-length APP and of APP
C-terminal fragments (CTFs) in total lysates (Fig. 5b), as well
as of soluble a-APP and b-APP in the extracellular medium
(Fig. 5c), are similar in 7PA2 and in 7PA2-A13K cells. Likewise,
in comparative subcellular fractionation studies, the scFvA13KDEL intrabody does not affect the intracellular maturation and
processing of APP, assessed by evaluating the compartmentspecific (ER versus Golgi) N- and O-glycosylation of APP and the
CTFs levels (Fig. 6a). Finally, the subcellular activities of a- b- and
g-secretase were found not to be affected by the scFvA13-KDEL
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b
Lumen/extracellular space
s-α-APP
s-β-APP
Total lysate
APP
Aβ
mAPP
iAPP
C99
C89 APP-CTFs
C83
β
α
γ
γ
C-83
AICD
C-89/99
β-Actin
AICD
Cytosol
mAb 6E10 (Aβ 3-9)
pAb α-APP C-term (last 20 aa)
mAb 4G8 (Aβ 17-24)
mAb 22C11 (APP 66-81)
A2
3K
7P -A1
2
A
7P
c
s-αAPP
+
Levels of APP species
(% of 7PA2)
s-αAPP
7PA2
7PA2
7PA2-A13K
120
100
80
60
40
20
0
mAPP
120
Conditioned
media
Levels of APP species (% of 7PA2)
a
iAPP
C99
C89
C83
AICD
7PA2-A13K
100
80
60
40
20
s-βAPP
A2 13K
7P
-A
A2
7P
0
s-αAPP
s-αAPP
+
s-βAPP
Figure 5 | Effects of the intrabody on the expression, maturation and processing of APP. (a) Scheme of APP fragments generated during APP–RIP
and epitopes recognized by different anti-Ab/APP antibodies used. (b) WB (anti-APP C-terminal) for full-length APP and for APP–CTFs in 7PA2
and in 7PA2-A13K lysates. i-APP and m-APP indicate the immature (N-glycosylated) and mature (N þ O-glycosylated) forms of APP. The APP–CTFs
are: C83, deriving from a-, C89 and C99 from b-secretase cleavage. The AICD derives from g-secretase activities (see a). The histogram shows
the levels of different APP forms in 7PA2-A13K lysates in comparison with 7PA2 cells (at 100) obtained from densitometric values of bands and
normalized versus b-Actin. Mean values±s.e.m., n ¼ 3. No significant differences between 7PA2 and 7PA2-A13K cells were observed. (c) WB
for soluble sa-APP (mAb 6E10) and sa-APP þ sb-APP (anti-APP N-terminal mAb 22C11) in 7PA2 and in 7PA2-A13K CM, deriving from a- and
b- secretase APP-cleavage. The histogram shows the levels of soluble APPs in 7PA2-A13K CM in comparison with 7PA2 CM (at 100) obtained from
densitometric values of bands and normalized as discussed in the method section and in the Supplementary Fig. 5. Mean values±s.e.m., n ¼ 3.
No significant changes of soluble APPs were observed in 7PA2-A13K cells in comparison with 7PA2 cells.
intrabody, as measured in comparative analyses of 7PA2 and
7PA2-A13K cells (Fig. 6b,c). In detail, the g-secretase activity was
assayed in subcellular ER-enriched fractions after their incubation
for 2 h at 37 °C, by measuring the selective reduction of the
APP–CTFs and the appearance of the APP intracellular domain
(AICD) (Fig. 6b) (as previously described31). Furthermore, we
established an assay to determine both g-secretase and a-,
b-secretases activities through a reversible block of intracellular
trafficking from ER to Golgi with Brefeldin A (BFA). One or four
hours of treatment with BFA determines: (i) a time-dependent
reduction of APP–CTFs (almost complete after 4 h of treatment)
indicative of g-secretase processing; (ii) a significant change in the
ratio of immature/mature APP forms, with a prevalence of the
immature one, indicative of the block of APP in the ER. After a
2 h washout of BFA: (i) the APP–CTFs are reestablished or
de novo generated (indicative of a-, b-secretases activities likely in
post-ER compartments); (ii) the maturation (glycosylation) of
full-length APP, previously prevented by the BFA treatment, is
restored (Fig. 6c).
All these assays show that in 7PA2 cells, the anti-AbOs
scFvA13-KDEL does not affect the maturation and processing of
APP, not even during its trafficking in the early secretory pathway
where the intrabody resides, shuttling between ER and cis-Golgi.
Remarkably, also in other cell lines (7WD4 and SHSY5Y) with
lower or negligible levels of AbOs, scFvA13-KDEL does not
interfere with the physiological APP processing (Supplementary
Fig. 9).
In conclusion, our data demonstrate that the scFvA13-KDEL
intrabody acts effectively and selectively on the levels of extraand intracellular pools of AbOs, with no interference with APP
expression, maturation or processing.
This molecular and subcellular selectivity represents a
significant experimental advance to modulate the levels of Ab
peptides, in comparison with widely used approaches of
g-secretase inhibition. Indeed, as expected, we found that in
7PA2 cells the g-secretase inhibitor L-685,458 (ref. 29)
dramatically decreases the production of Ab and, consequently,
of AbOs, but massively increases the levels of a- and b-CTFs,
altering also their subcellular distribution, and the levels of
soluble APPs (Supplementary Fig. 10).
The lack of interference of the intrabody on the APP cellular
biochemistry is noteworthy due to several functions of
APP-derived fragments (that is, of soluble APPs and of AICD)32.
ScFvA13-KDEL selectively binds AbOs in the ER. In order to
explore the selective mechanism of AbO modulation, we first
investigated the interaction of the intrabody with subcellularly
localized Ab/AbOs species, by microsome fractionation of
7PA2-A13K cells, followed by co-immunoprecipitation (co-IP)
and WB analyses. We demonstrated the intrabody-mediated
selective pulldown of LDS/SDS-stable Ab dimers, trimers and
small oligomers (o20 kDa), but not of Ab monomers, neither of
the full-length APP, in the ER-enriched fractions of 7PA2-A13K
NATURE COMMUNICATIONS | 5:3867 | DOI: 10.1038/ncomms4867 | www.nature.com/naturecommunications
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ARTICLE
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
Subcellular fractions
ER fraction
7PA2
7PA2-A13K
APP
mAPP
iAPP
APP-CTFs
APP-CTFs
AICD
Calnexin
Calnexin
ER Golgi
7PA2
4 °C 37 °C 4 °C 37 °C
7PA2
7PA2-A13K
ER Golgi
7PA2-A13K
2.5
Control (%)
2
Total lysates
7PA2-A13K
7PA2
1
0
BFA BFA (1 h) BFA BFA (4 h)
+
+
(1 h)
(4 h)
WO (2 h)
WO (2 h)
–
–
–
–
–
+
–
+
+
+
–
+
+
–
–
–
–
–
–
+
–
BFA 1 h
–
–
+
+
–
+
–
+
–
–
BFA 4 h
–
+
Wash-out
(WO) 2 h
Control (%)
β-Actin
–
7PA2-A13K
1.5
APP-CTFs
+
APP-CTFs levels
7PA2
0.5
mAPP
iAPP
+
–1
0
1 2
Variations at 37 °C
1.4
1.2
1
0.8
0.6
0.4
0.2
0
mAPP/iAPP
7PA2
7PA2-A13K
BFA BFA (1h) BFA BFA (4 h)
+
+
(4 h)
(1 h)
WO (2 h)
WO (2 h)
Figure 6 | The scFvA13-KDEL does not alter the subcellular maturation and processing of APP. (a) WB analysis (anti-APP C-terminal) of the
compartment-specific APP maturation and processing in representative subcellular fractions (ER or Golgi enriched) of 7PA2 and 7PA2-A13K cells. The ER
and Golgi specificities (such as the different ratio i-APP/m-APP, and the different CTFs levels) are similar in 7PA2 and 7PA2-A13K cells. (b) g-secretase
activity in subcellular ER-enriched fractions of 7PA2 and 7PA2-A13K cells assayed by WB (anti-APP C-terminal). Freshly prepared ER-enriched fractions are
incubated for 2 h at 37 °C or at 4 °C, to allow (37 °C) or not (4 °C) specific g-secretases APP cleavages at the g- and E-sites, respectively measured by the
selective reduction of the APP–CTFs C99-C83 (but not of the full-length APP) and by the appearance of the AICD. The histogram shows the variations at
37 °C of APP, APP–CTFs and AICD (densitometric values of WB bands normalized versus calnexin). Mean values±s.e.m., n ¼ 3. No significant differences
between 7PA2 and 7PA2-A13K cells were observed. (c) Total cell lysates WB analysis of APP and APP–CTFs (anti-APP C-terminal), in experiments of
reversible block of intracellular trafficking from ER to Golgi complex: transient (1 or 4 h) BFA treatment induces a reduction of APP–CTFs, which can
be completely reversed after a 2 h BFA washout. The reduction of APP–CTFs is indicative of g-secretase processing, whereas the re-establishment (or de
novo generation) of CTFs levels is indicative of a-, b-secretases activities. The upper histogram shows the variations at of APP–CTFs (densitometric values
of WB bands normalized versus b-Actin, and represented as % of corresponding control). Mean values±s.e.m., n ¼ 3. No significant differences between
7PA2 and 7PA2-A13K cells were observed. In addition, as expected, 1 or 4 h BFA treatment blocks also the maturation of the full-length APP. The
lower histogram shows the variations of the ratios m-APP/iAPP (calculated from densitometric values of WB bands normalized versus b-Actin, and
represented as % of corresponding control). Mean values±s.e.m., n ¼ 3. No significant differences between 7PA2 and 7PA2-A13K cells were observed.
cells (Fig. 7a). The AbO pulldown was successful when freshly
prepared microsome vesicles were subjected to a preliminary step
of chemical cross-linking; this suggests a transient nature of the
AbO-intrabody complexes (consistent with a chaperone-like
interaction of the intrabody).
The binding selectivity of scFvA13-KDEL intrabody for AbOs
in living cells, with respect to the monomeric Ab, is in accordance
with the in vitro oligomer-selective discrimination and conformation binding properties of the scFvA13 (ref. 13), but the fact
that we could prove it in the intracellular environment is
noteworthy. Furthermore, the binding selectivity with respect to
the APP further suggests that, in the ER lumen, the Ab moiety in
the context of APP is not recognized by the scFvA13-KDEL
intrabody, in line with the lack of interference with APP
processing and maturation (Figs 5,6).
We suggest that the primary action of scFvA13-KDEL
intrabody is to bind to early-assembled, possibly transient,
AbO conformers (here detected in the co-IP/WB analysis as
8
SDS/LDS-stable Ab dimers and trimers and small AbOs), but not
to the free Ab monomers. This could inhibit a further Ab
oligomerization, most likely due to the interference with critical
seeding defined by the bound AbO conformers. The significant
reduction of intracellular pool of AbOs measured by A11
antibody (see Fig. 4), which preferentially recognizes prefibrillar
AbOs larger than dimers and trimers30, is also in accordance with
this proposed mechanism.
The co-IP results (Fig. 7a) and the DB immunoreactivity of cell
lysates with A11, scFvA13 and scFvIm3 (Fig. 4) (the latter
confirmed as Ab-specific by the g-secretase inhibitor treatment
(Supplementary Fig. 7b)), demonstrate that AbOs larger than
dimers and trimers exist in the intracellular environment of 7PA2
cells. We conclude that the intrabody-mediated interference in
the ER with the dynamic pool of intracellular AbOs, including
oligomers larger than Ab dimers and trimers, can influence also
the balance of extracellular AbOs, which are mainly detected as
LDS/SDS-stable dimers and trimers.
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a
b
Input
IP
scFvA13-KDEL
*
20 kDa
30 kDa
15 kDa
10 kDa
3.5 kDa
ER fraction
7PA2
ER fraction
7PA2-A13K
Aβ trimers
20 kDa
Aβ dimers
15 kDa
Aβ
10 kDa
7PA2 CM
*
* *
200
50 kDa
40 kDa
APP
*
110 kDa
80 kDa
60 kDa
Densitometric values (a.u.)
Input IP
3.5 kDa
+
–
–
–
+
+
–
–
+
+
+
–
+
+
–
+
Aβ1-42 (65 μM)
24 h aggregation
scFvA13 (13 μM)
scFvA13 (65 μM)
150
100
50
0
4 kDa
8 kDa
10-20 kDa 30-80 kDa
Figure 7 | Intrabody binding of AbOs in the ER and interference of scFvA13 with Ab assembly in vitro. (a) Intrabody-mediated selective pulldown
of AbOs: co-IP of scFvA13-KDEL intrabody with Ab dimers, Ab trimers and larger AbOs (o20 kDa) but not with Ab monomers nor APP, from an
ER-enriched fraction of 7PA2-A13K cells (7PA2 cells as intrabody negative control). The co-IP was obtained by an anti-V5 coupled resin, to bind the
C-terminal V5 tag of the intrabody and blotted to detect the different Ab species and APP (mAb 6E10) and for the intrabody (anti-V5). * indicates the
light chain of the anti-V5 IP antibody. On the right, representative Ab bands detected (by 6E10-WB) in 7PA2 CM in the 3.5–15 kDa range. (b) WB analysis
(rabbit mAb anti-Ab) of Ab assembled in vitro with or without scFvA13. The human Ab1-42 peptide (65 mM) was aggregated at 22 °C for 24 h
without scFvA13, or in the presence of substoichiometric (13 mM, ratio Ab:scFv, 1:5) or stoichiometric concentrations (65 mM) of scFvA13. According
to previous reports47, after the in vitro assembly, synthetic Ab1-42 shows a pattern of HMW oligomers (30–80 kDa) absent at the early steps of
aggregation (in which Ab monomers and LMW oligomers (o20 kDa) prevail). Of note, the co-presence of LMW oligomers and monomers cannot
be ruled out in starting solutions of synthetic Ab70, being these species in rapid equilibrium and determining variability on the levels of Ab monomers
measured. Both sub- and stoichiometric concentrations of scFvA13 during the assembly of Ab1-42 determine the following: (i) the lack of formation of
HMW AbOs; (ii) a pattern of LMW AbOs similar to that at the early steps of aggregation. This shows that the scFvA13 interferes with critical
conformational changes during the early assembly, avoiding the formation of larger AbOs. The histogram shows the variations of Ab species obtained
from densitometric analysis. Mean values±s.e.m., n ¼ 3; *Po0.01 (Student’s t-test).
The scFvA13 interferes with AbOs assembly in vitro. We
studied next the effects of scFvA13 (used as purified protein) on
the formation of AbOs or on preformed AbOs in vitro. Indeed,
while considering obvious and well-known differences between
natural and synthetic Ab samples (that is, the high concentration
of synthetic Ab used in vitro and the different appearance of Ab
assemblies)33 such in vitro study could help interpreting the
mechanism of conformational interference of scFvA13-KDEL
intrabody. We demonstrated that the scFvA13, both at
stoichiometric and substoichiometric ratios with the Ab1-42
monomeric peptide, strongly inhibits the assembly of AbOs
in vitro. Indeed, in the presence of scFvA13, we observed the lack
of formation of high-molecular weight (HMW) AbOs (30–80 kDa
in WB analysis) from a starting solution where Ab monomers
and low-molecular weight (LMW) oligomers (o20 kDa) are in
rapid equilibrium (Fig. 7b). Thus, the scFvA13 blocks the
assembly from LMW towards HMW AbOs (Fig. 7b). In
addition, we studied whether and how the scFvA13 acts on
preformed AbOs. When incubated with scFvA13, LMW and
HMW AbOs undergo a change of their assembly state, as
revealed by the reduction of their MWs with a concomitant
significant increase of free Ab monomers (in a scFv
concentration-dependent manner) (Supplementary Fig. 11).
Both these in vitro observations (dynamic interference in
assembly and disassembly pathways) support our hypothesis on
the mechanism of action of the scFvA13-KDEL intrabody on
AbO conformers and are in accordance with the binding and
pulldown into the ER and with measured overall modulation of
Ab/AbOs levels in cells (reduction of AbOs and the increase of
secreted Ab monomers).
Intracellular fate of scFvA13-KDEL. In order to support the
mechanism by which the scFvA13-KDEL intrabody intercepts
early AbO conformers in the ER and acts as a conformationalsensitive and chaperon-like assembly modulator, we better
characterized the subcellular localization of scFvA13-KDEL and
its possible delivery to pathways of cellular degradation.
We found that the intrabody does not localize in trans-Golgi
network (TGN), neither in early nor late endosomes and in
lysosomes (as demonstrated by indirect immunofluorescence, by
the lack of co-localization with the subcellular markers TGN46,
Rab5, Rab7 or LAMP-1, respectively) (Fig. 8a). Thus, the
intrabody, as expected for a physiological and non-altered KDEL
system, is mainly restricted into the ER excluding its localization
in other endocytic vesicles which are crucially involved in
recycling or degradation pathways. Of note, the TGN was
recently identified as a crucial compartment for the synthesis of
Ab40 mainly due to the APP re-routing from the plasma
membrane via the retromeric pathway34. Also endosomal,
lysosomal and the secretory pathway (ER, Golgi) were
demonstrated to be sites of Ab production7. However, the
precise localization of the scFvA13-KDEL in the ER (and, only
partially, with cis-Golgi), as demonstrated, strongly support the
view of a more confined action of the intrabody in these
compartments. This formally establishes the ER (and cis-Golgi) as
a major organelle where the control of Ab oligomerization is
crucial.
We next investigated if the scFvA13-KDEL intrabody undergoes possible mechanisms of ER-associated degradation (ERAD)
that acts through the ER to cytosol retrotranslocation of
ER-resident proteins and their degradation by targeting the
proteasome35. In principle, the ER-localized intrabody might
escort the bound AbOs to the ERAD pathway. We observed,
however, that in 7PA2-A13K cells, the levels of the intrabody do
not change after 6-h treatment with the proteasome inhibitor
MG132 (20 mM) (Fig. 8b). On the other hand, in 7PA2 cells stably
expressing a different version of the intrabody, referred to as
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ARTICLE
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a
scFvA13-KDEL
Subcellular marker
Merge
TGN46
Rab5
Rab7
b
scFvA13-KDEL
β-Actin
MG132
CHX
–
–
+
–
–
+
Densitometric values
(normalized)
LAMP-1
2
1.5
1
0.5
*
0
Control
MG132
CHX
Figure 8 | The subcellular fate of scFvA13-KDEL intrabody. (a) Confocal analysis of 7PA2-A13K cells. The localization of scFvA13-KDEL (detected by
using an anti-V5, see Fig. 2a) is showed in the left panels. The middle panels show the localization of different subcellular markers, from the top TGN46
(for the trans-Golgi network), Rab5 (for early endosomes), Rab7 (for late endosomes) and LAMP-1 (for lysosomes). The panels on the right show the
respective merged images. Scale bar, 10 mm, magnification 63. No significant co-localization of the ER-resident intrabody with any subcellular marker was
observed, excluding mechanisms of mis- or re-localization of scFvA13-KDEL from the ER, or its sorting in recycling (endosomes-TGN) or degradation
(lysosomes) pathways. (b) WB analysis (anti-V5 tag) of scFvA13-KDEL in 7PA2-A13K cells after 6 h MG132 (20 mM) treatment (to block the proteasome
and study the ERAD pathway) or after 6 h cycloheximide (CHX) (40 mM) to block the protein synthesis. The histogram shows the levels of scFvA13-KDEL
in the different treatments (values normalized from densitometric analysis of bands by ScionImage software). No significant changes in MG132 treatment
were observed, excluding ERAD mechanisms. Comparative experiments show that a different version of the intrabody, referred to as scFvA13-degradin,
undergoes significant ERAD (Supplementary Fig. 12). Mean values±s.e.m., n ¼ 3; *Po0.01 (Student’s t-test) in CHX treatment both versus control and
versus MG132 treatment.
‘scFvA13-degradin’ (specifically targeted to the ERAD system via
C-terminal fusion with the moiety of SEL1L protein involved in
the retrotranslocation process)36, we observed a significant
10
increase of the intrabody levels after 6 h MG132 treatment
(Supplementary Fig. 12). Thus, the scFvA13-degradin undergoes
ERAD and proteasome degradation, while the scFvA13-KDEL
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does not. As control, a 6-h treatment with cycloheximide
(blocking the protein synthesis) dramatically reduces the levels
of both scFvA13-KDEL (Fig. 8b) and scFvA13-degradin
(Supplementary Fig. 12) suggesting for both intrabody forms a
short yet similar half-life. The short half-life of the intrabody
scFvA13-KDEL is also confirmed by an in vitro assay on
ER-enriched subcellular fractions, where the intrabody
undergoes an almost complete degradation after 6 h of
incubation at 37 °C (Supplementary Fig. 13).
The overall data suggest that the scFvA13-KDEL is mainly
located in the ER, does not alter the APP maturation and
processing, and does not undergo ERAD process. Furthermore,
the short half-life of scFvA13-KDEL, together with its moderate
levels of expression, prevents its accumulation in the ER and a
consequent ER stress. We conclude that observed variations of
AbO levels and the overall balance of secreted Ab species are
representative of a fine mechanism of conformational assembly
modulation of AbO, which occurs in the ER (and in KDEL
recycling compartments) where the intrabody intercepts critical
conformers changing their oligomerizing fate, rather than their
targeted degradation to proteasome or their subcellular
re-targeting in other active endocytic pathways.
Functional consequences of the intrabody interference with
AbOs. In order to investigate the functional consequences of the
selective interference of the scFvA13-KDEL intrabody with
specific pools of AbO conformers, we exploited two established
experimental readouts on 7PA2 cells. The AbOs secreted by 7PA2
cells induce specific AD-related synaptic dysfunctions, when
administered to primary neurons, brain slices or rodent
brains3,8,9,24–26. In detail, in the primary hippocampal neurons,
AbOs (contained in 7PA2 CM) can induce an impairment of the
signalling mediated by extrasynaptic NR2B-containing NMDA
receptors, modulating the ERK1/2 and CREB phosphorylation
status26. Here, we demonstrate, in this experimental paradigm,
that both ERK1/2 and CREB are largely dephosphorylated in the
hippocampal neurons treated with 7PA2 CM but significantly less
in those treated with 7PA2-A13K CM (Fig. 9a). This provides a
functional demonstration that the AbO conformers targeted by
the scFvA13-KDEL (and selectively immunodepleted in the
7PA2-A13K CM) are involved in the activation of a NR2BNMDA extrasynaptic signalling, which is implicated in
neurodegenerative cascades37.
Furthermore, we reported an independent functional demonstration of the biological relevance of the intrabody-targeted AbO
conformers. Indeed, in 7PA2 cells, the activity of mTOR
(mammalian target of rapamycin), a crucial mediator of cellular
homeostasis and autophagy, is directly linked to the levels of
endogenous Ab38. Here, we show that in 7PA2-A13K cells,
mTOR activity is reduced back to basal levels (found in CHO
cells), as measured by the phosphorylation levels of the direct
downstream target p70S6K (Fig. 9b). This provides a new
functional demonstration that the intracellular AbO conformers
targeted by the scFvA13-KDEL are involved in the impaired
homeostasis and autophagy in 7PA2 cells, through the mTOR
signalling pathway.
In conclusion, the functional results, through two independent
readouts, demonstrate that both extra- and intracellular pools of
AbO conformers targeted by the scFvA13-KDEL intrabody,
among the numerous and diverse Ab and APP-derived soluble
species, constitute a crucial pool of active AbOs, directly involved
both in the deregulation of specific cellular pathways linked to
cellular homeostasis, and in the synaptic actions previously
ascribed to the more complex CM of 7PA2 cells, and thought to
be relevant in neurodegeneration and also AD pathogenesis.
Discussion
AbOs are considered the most neurotoxic Ab forms in AD brains,
but they are still mysterious entities in terms of molecular
and structural composition and activity8. Furthermore, while
intracellular AbOs are emerging targets in AD studies7,15, their
identification, study and targeting in the complex context of
subcellular compartments of living cells still remains a big
challenge. This is a key issue, in order to uncover and validate
new molecular and cellular targets tightly linked to the Ab
hypothesis, for a successful search and development of novel
therapeutics for AD8,39.
While the processes of Ab aggregation are intensively studied
at different levels of complexity (in silico, in vitro and in vivo)
numberless questions remain open and to be addressed, such as
the identification of the cellular mechanisms and the subcellular
sites of Ab misfolding and oligomerization. Of note, in the
current state of the art, this question necessarily calls for
the development of new selective experimental approaches
exploitable in living cells.
We face the question in a way not tackled before by
establishing a new approach that we called CSI based on the
subcellular targeting of a conformation-sensitive intrabody in
living cells. In this way, we showed for the first time that it is
possible to selectively target, inside the cell, certain oligomeric Ab
conformers but not the Ab precursor protein (APP) or the
monomeric Ab. This represents a significant advancement with
respect to the currently used secretase inhibitors40 and previous
targeting of the Ab/APP system with intrabodies that were acting
through an interference with the processing and/or maturation of
APP41 or of g-secretase components such as nicastrin42.
Moreover, the subcellular-localized anti-AbOs CSI, via the
retention/retrieval of the KDEL intrabody in the ER, provides
significant new insights into the functions of this organelle in the
assembly of functionally active AbOs.
We show that the main mechanism operated by the ERresident intrabody is of a conformational assembly modulation,
through a mechanism of chaperone-like folding assistance on
critical AbO conformers intermediates (Fig. 10).
The CSI determines an opposite balance of the different pools
of soluble Ab observed in 7PA2-A13K cells: on one hand, the
extracellular LDS/SDS-stable dimers and trimers and the AbO
conformers are reduced, and, on the other hand, the extracellular
monomeric Ab peptides are increased, without significant
changes in the ratios of Ab truncated forms.
Recent studies show the AD pathological importance of
qualitative changes in the g-secretase-generated Ab products
(species of 37–43 amino-acid length)43. Even small changes in the
composition of synthetic Ab mixes can affect critically their
aggregation kinetics and toxic effects; in particular, AbOs
composed by fAD-like Ab42:Ab40 ratios are more stable and
toxic44. This is in accordance with the differences observed here,
between 7PA2 and 7WD4 cells. However, our results in 7PA2A13K cells (where we observed complementary changes of the
secreted AbOs and of Ab monomers/fragments) additionally
show that the ER control of Ab peptides folding and assembly
in vivo, even in the context of their pathological ratios, is a crucial
step in the formation of pathologically relevant AbO conformers.
In 7PA2-A13K cells, the increased proportion of secreted
free monomers can be interpreted as deriving from the
intracellular AbO conformers intercepted by the intrabody along
oligomerization pathways and representing a fingerprint of their
composition (in non-assembled state). Thus, AbOs, when
assembled inside the cells and then secreted, would be formed
by well-defined ratios of several Ab peptides and fragments.
The Ab1-42, known to be preferentially generated in the
ER7,45,46, could act here as a seed for oligomerization of other Ab
NATURE COMMUNICATIONS | 5:3867 | DOI: 10.1038/ncomms4867 | www.nature.com/naturecommunications
& 2014 Macmillan Publishers Limited. All rights reserved.
11
ARTICLE
M
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
U
nt
re
a
7P ted
A2
C
M
7P
A2
-A
13
K
C
a
pCREB (phospho-Ser133)
pERK1/2 (phospho-Thr202/Tyr204)
7PA2 CM
ERK1/2
Phosphorylation
(normalized)
1.2
7PA2-A13K CM
*
*
1
0.8
pERK1/2
0.6
pCREB
0.4
0.2
M
K
C
C
M
*
β-Actin
p70S6K (total)
2.5
2
1.5
1
0.5
p70S6K (phospho-Thr389)
0
7P
A2
-A
13
7P
A2
β-Actin
C
H
O
mTOR (phospho-Ser2448)
*
K
mTOR (total)
p70 phosphorylation
3
7P
7P
A2
b
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-A
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Figure 9 | Functional interference knockdown of AbOs by scFvA13-KDEL. (a) On the left, a simplified scheme of the experiment in which the
CM from 7PA2 or 7PA2-A13K cells is administered to the primary hippocampal neurons. On the right, WB analysis of ERK1/2, phospho-ERK1/2
(pThr202/pTyr204) and phospho-CREB (pSer133) of the hippocampal neurons treated with 7PA2 CM and 7PA2-A13K CM for 16 h, and histogram of the
phosphorylation ratios from densitometric analysis values. Mean values±s.e.m., *Po0.05 Student’s t-test, n ¼ 3. The 7PA2-A13K CM is effectively
depleted of functionally active AbOs by the intrabody-mediated CSI. The modulation of the ERK and CREB signalling pathways by AbOs, mediated by
NR2B stimulation, was preliminarily verified by the use of the NR2B-selective antagonist Ro 25-6981 (0.5 mM) (unpublished data) according to the
previous reports26. (b) WB (steady-state levels) of mTOR, phospho-mTOR (pSer2448), p70S6K and phospho-p70S6K (pThr389) in 7PA2,
7PA2-A13K and CHO cells and histogram of the p70S6K phosphorylation ratios from densitometric analysis values. Mean values±s.e.m.,
*Po0.05 Student’s t-test, n ¼ 3. The scFvA13-KDEL intrabody, through its functional knockdown of AbOs, reduces mTOR activity.
monomers with a mechanism therefore favored by the neutral
luminal pH (peculiar of the ER) and by the macromolecular
crowding of this organelle. Of note, in vitro studies demonstrate
that neutral pH, opposite to acid pH, favors pathways of
Ab oligomerization against fibrillization47 and that the
macromolecular crowding can contribute to the amyloid
aggregation48.
Besides its favourable physicochemical milieu, the ER has a
crucial role in the protein quality control and in the physiological
oligomerization of some protein complexes, making a priori this
organelle a good candidate for our intrabody-targeting study. The
chaperone-like mechanism proposed for the scFvA13-KDEL is
consistent with the physiological role of ER-resident chaperones
engaged in protein folding and/or degradation processes35.
However, it is unknown if AbOs can be a direct target of
endogenous ER chaperones; a study demonstrated that some ER
chaperones indirectly regulate the Ab production through their
interaction with APP impairing its maturation and processing49.
Thus, the scFvA13-KDEL intrabody can equip the ER with new
beneficial functions (otherwise lacking in mammalian cells) of
12
selective control of critical AbO assembly. We also surmise that
the KDEL system, physiologically exploited by some endogenous
ER chaperones for the protein quality control, was likely essential
to obtain a chaperone-like action of the intrabody (shuttling
between the ER and the Golgi complex).
Several studies show that the Ab peptides can be formed in
different subcellular compartments such as ER, Golgi, TGN,
endosomes, lysosomes7,34 but no study has been able to address
the important question of where and how intracellular Ab
peptides oligomerize. Recently, intracellular AbOs have been
identified in fixed cells or brain tissues through oligomericspecific antibodies, but these antibodies do not allow functional
studies of the AbOs formation and trafficking in living cells.
Thus, the localization of intracellular AbOs is still an open and
controversial question; as for the origin of intracellular AbOs, the
prevailing hypothesis suggests the endocytosis of AbOs assembled
outside of the cell. However, the extracellular space, such as the
extracellular fluid of the brain, is not the ideal milieu for the Ab
aggregation, first due to the very low concentration of soluble Ab
(o1 nM). Accordingly, a study demonstrated that nanomolar
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
Aβ
Secretases inhibitors
ER
KDEL-R
Aβ
APP
Secretory
AβOs
Aβ
Endocytic vesicles
scFvA13-KDEL
AβOs
Aβ AβOs
AβOs
7PA2
Aβ
KDEL-R
ER
Aβ
AβOs
Secretory
scFvA13-KDEL
AβOs
Endocytic vesicles
Aβ AβOs
7PA2-A13K
Figure 10 | Model of mechanism of action of the intrabody-mediated CSI for scFvA13-KDEL. The panels are based on the scheme of Fig. 1b representing
the APP processing and Ab generation and oligomerization in the cellular model of APP–RIP used in the study. (a) Proposed mechanism of action of
scFvA13-KDEL in the pathway of Ab generation and assembly: while secretase inhibitors target the APP cleavages, the intrabody controls the assembly of
Ab in pathological AbOs without interfering with the APP processing. (b) Schematic 7PA2 cell used as fAD model of APP–RIP and AbOs generation.
Our study demonstrates that the ER is an organelle crucial for the assembly of biologically relevant AbOs. The ER-assembled AbOs are likely the main
source of those found extracellularly and they are essential in the intra- and extracellular balance of Ab and AbO species. (c) In 7PA2-A13K cells, we
propose a mechanism by which early-assembled AbO conformers are intercepted by the intrabody in the ER. Thus, the intrabody can be deputed to a
selective control of the Ab misfolding (represented by a change of the green–red-shaded box with a green one) and assembly (represented by smaller
red frame boxes in comparison with 7PA2 cells), equipping the ER with new functions of quality control, otherwise escaped by pathological conformations
of Ab. Thus, the main mechanism operated by the ER-resident intrabody is of a conformational assembly modulation. As a consequence, the levels of
intra- and extracellular pools of AbOs decrease while secreted Ab monomers increase.
concentrations of synthetic Ab, extracellularly administered to
the cultured cells, form amyloid seeds only in the intracellular
milieu upon cellular uptake, concentration and aggregation in
endocytic vesicles50. At difference, our study is performed
in living cells where a complex pattern of Ab peptides is
endogenously produced through the APP–RIP occurring in
several subcellular compartments. In this context, we
recognized the ER as the main and crucial site to control
critical events of Ab oligomerization, just exploiting the precise
spatially localized subcellular intrabody probe for CSI (in the ER
but not in other endocytic vesicles). However, we cannot exclude
that some Ab oligomerization (or Ab super assembly onto
ER-formed seeding nuclei) could also occur in other subcellular
compartments (that is, along the secretory and endocytic
pathways), and/or that some Ab/AbO species can reach the ER
through the Golgi complex, during retrograde routes of
subcellular vesicular trafficking that is, via the retromer TGN).
Finally, we demonstrated that the pool of AbO conformers
intercepted in the ER and modulated by the scFvA13-KDEL
intrabody is functionally relevant, by exploiting two distinct
functional readouts regarding the extrasynaptic activation of
ERK/CREB signalling pathways in primary neurons and the
phosphorylation pathway of p70S6K, a downstream target of
mTOR, implicated in the impaired cellular homeostasis and
autophagy. As an interesting parallel to our results, amyloidbinding compounds modulating the Ab assembly also restored
the protein homeostasis during aging in Caenorhabditis elegans
models51. Given the central role of the ER mitochondria crosstalk in maintaining cellular homeostasis, and given that 7PA2
cells show a prominent set of functional mitochondrial deficits52,
it will be interesting to investigate whether the scFvA13-KDEL is
able to restore normal mitochondrial functions in 7PA2 cells, via
its interference with intracellular AbOs in the ER.
Our overall results support the idea that targeting specific
conformations of Ab can be a strategy more selective and more
effective than lowering its total levels. Furthermore, we show the
value of the subcellular targeting for mechanistic studies on Ab
oligomerization in the ER and the early secretory compartment.
In addition, intrabodies can be targeted to other subcellular
compartments and also to neuronal specialized districts in order
to address new relevant questions on different mechanisms of
AbOs trafficking and actions. Indeed, the cellular mechanisms of
APP processing, Ab generation and oligomerization can present
specific cell-type peculiarities, as in the case of the polarized and
specialized neuronal cells. In neurons, APP undergoes fast
anterograde transport to nerve terminals and is metabolized
within the axonal or presynaptic vesicles into Ab peptides that are
released and deposited as amyloid plaques around nerve
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867
terminals53,54. Furthermore, synaptic activity increases Ab
secretion, indicating that the presynaptic terminal is an
important regulatory site for Ab generation55,56. Of note,
intrabodies can be targeted also to neuronal specialized
districts, such as pre- and post-synaptic terminals57,58. The
demonstration of cell-specific (neuronal or not) mechanisms of
subcellular AbOs trafficking and actions will be the next goal of
our intrabody-based studies, having here established that CSI
allows a dissection of AbOs-specific mechanisms not mediated by
Ab monomers nor by APP.
In light of the controversial and open questions emerging from
Ab-targeting clinical trials39 and immunotherapy59 in AD, we
believe that our study provides a new cellular and biochemical
basis for targeting AbOs inside cells, and that the CSI approach
can be worthy of further preclinical and clinical applications. Of
note, the conformation selectivity and the molecular specificity of
the CSI approach overcome limits of methods targeting nucleic
acid (genes or mRNA) or exploiting new chemical compounds
that cannot achieve a comparable molecular and subcellular
precision. Finally, the CSI approach can be exploited in different
proteinopathies through the upstream direct selection of new
functional intrabodies60,61 against antigens that undergo in vivo
misfolding, similarly to Ab13.
Methods
Cell lines. 7PA2 and 7WD4 cells (CHO stably transfected respectively with the
human APP751 cDNA bearing the V717F fAD mutation and with the wtAPP751,
kindly provided by Dr D.J. Selkoe, Harvard Medical School, Boston) were grown in
Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS),
2 mM glutamine and antibiotic selection (G418) as previously described23. SHSY5Y
human neuroblastoma cells were grown in DMEM-F12 supplemented with 10%
FBS and 2 mM glutamine.
Intrabody constructs. The constructs for scFv-KDEL intrabodies for the ER
retention/retrieval, follow the scheme of expression box of the pscFvExpress
vector62, with an N-terminal SecL, secretory leader sequence from an IgG1
immunoglobulin, and a C-terminal KDEL, ER retention/retrieval signal sequence.
A V5 tag is inserted before the C-terminal KDEL sequence and allows the
immunodetection of the scFvs, by anti-V5 antibodies. The cDNA of the anti-AbOs
scFvA13 (ref. 13) (or of the Ab-unrelated scFvR4 (ref. 63)) was cloned through the
restriction enzyme BssHII-NheI in frame with a C-terminal V5 tag. The secretory
construct differs from the KDEL construct only for the lack of the C-terminal
KDEL ER retention/retrieval signal sequence. The cDNA of scFvA13 was also
cloned in the backbone vector ‘scFv-degradin’ (kind gift of Prof. Burrone, IGEB
Trieste)36.
Establishment of cell lines stably expressing the intrabodies. Cells were
transfected with scFv-intrabody vectors, coding for scFvA13 or scFvR4, using
Lipofectamine 2000 (Invitrogen) and stable cell clones were obtained by limiting
dilution in the presence of appropriate antibiotics (250 mg ml 1 G418 and
2.5 mg ml 1 puromycin for 7PA2 and 7WD4 cells, and only G418 for SHSY5Y
cells).
We established both a polyclonal population and multiple stable single clones.
In a preliminary survey, we characterized the levels of intrabody expression
(by WB), its subcellular localization (by immunofluorescence) and the modulation
of secreted AbOs. Then we performed the overall study on a 7PA2-A13K clone,
showing an average expression level and representative of the entire polyclonal
population.
Cell samples for Ab/AbOs analyses. For CM preparation, cells were grown to
B90% confluence, washed twice with Dulbecco’s phosphate-buffered saline
(DPBS) and then conditioned in serum-free medium for 18 h. CM was collected
and cleared of cell debris by centrifugation at 200g for 10 min. In comparative
analysis of different cell lines, CM was prepared from cells at the same density of
plating. The Ponceau S staining of nitrocellulose membrane before WB (detecting
the total proteins) and the WB for the extracellular soluble protein Laminin
(unpublished data) were used as normalization of WB lanes loading (see also
Supplementary Information).
In straight (non-concentrated) 7PA2 CM, Ab monomers are detectable (by
WB and SELDI-TOF MS), while AbOs (by WB and DB) are generally under
the detection limits. In order to improve the detection of AbOs (without
immunoprecipitation steps), we optimized a procedure of concentration of the CM
by centrifugation steps in concentrators with a 3 kDa or a 5 kDa cut-off (VivaSpin
14
Sartorius). We established that a 15 concentration allows a sensitive
discrimination of AbOs (in comparative analyses of 7PA2 and 7PA2-A13K CM).
The overall results demonstrate that this procedure is useful to obtain samples,
whose concentrations of total Ab (determined by quantitative WB) are in the range
of previous studies, and, most importantly, maintaining the effective
conformational-relevant differences of starting non-concentrated samples.
Cell lysates were done in Tris-HCl pH8 50 mM, NaCl 150 mM, NP40 1% with
the addition of cocktail of proteases and phosphatases inhibitors (Roche). The
lysates were centrifuged at 12,000g for 10 min. The supernatant was collected and
the concentration of total proteins was determined by BCA assay.
Ab/AbOs WB analysis. Samples (CM or cell lysates) were diluted in 4 LDS
sample buffer, containing reducing agents (DTT or b-mercaptoethanol), boiled
10 min, loaded in precasted Criterion gels XT Bis-Tris 4–12% Bio-Rad, running in
MES buffer. The semidry blot was then done onto nitrocellulose membrane filters,
0.22 mm. The membrane filter was boiled in PBS to increase the detection of low
MW bands. MAb 6E10 (Covance) or mAb 4G8 (Covance) were used, respectively
1:1,000 or 1:500 in TBS 0.05% tween, 2% dry milk. ECL (GE) chemiluminescent
detection was performed.
As discussed in the text, the bands at B8 kDa and at B12 kDa correspond to
Ab dimers and Ab trimers, collectively named LDS/SDS-stable low MW AbOs
(due to the analysis method involving LDS in the sample buffer and SDS in the
polyacrylamide gel), largely characterized in 7PA2 cells24. The identification of the
4, 8 and 12 kDa bands as containing Ab was confirmed by the use of a g-secretase
inhibitor L-685,458 (ref. 29), which almost abolishes the formation of Ab, and, as a
consequence, also of its assemblies, AbOs (see also Supplementary Information).
DB analysis. Purified proteins and cellular samples were spotted onto nitrocellulose membrane filters 0.22 mm. The anti-AbOs scFvA13 (3.5 mg ml 1) and
scFvIm3 (4.5 mg ml 1) were used as primary antibodies diluted in TBS 0.05%
tween, 5% dry milk with the addition of cocktail of proteases inhibitors (Roche).
The polyclonal antibody anti-oligomer A11 (ref. 21) was used 1:500 in TBS 0.05%
tween, 5% dry milk. The AbO scFv-positive immunodetection was performed by
anti-His tag (for cellular samples), or by anti-V5 tag (for monomers and oligomers
of synthetic Ab, a-Synuclein and lysozyme), which respectively recognizes the
C-terminal 6xHis tag or V5 tag of recombinant scFvs (expressed in Escherichia coli
and purified, see the paragraph below). Serial dilution curves of cellular samples
were preliminarily tested to obtain non-saturating condition of immunodetection.
Samples were loaded as follows: 450 ng per dot of synthetic human Ab1-42; 500 ng
per dot of recombinant human a-Synuclein; 500 ng per dot of lysozyme; 20 ml per
dot of 15 concentrated 7PA2 CM and related samples (CM of g-secretase
inhibitor-treated 7PA2 cells and 7PA2-A13K CM) by using Bio-Dot SF apparatus
Bio-Rad; 500 ng per dot of total lysates (non-denatured).
Purified proteins assayed by DB and ELISA. Synthetic human Ab1-42
(HFIP-pretreated peptide, purchased from Anaspec) was dissolved (5 mM) in
anhydrous DMSO and assembled to form AbOs as ADDLs (100 mM Ab in phenol
red-free Ham’s F12 medium, purchased from PanBiotech) at 4 °C for 24 h47
without shaking. Following incubation, tubes were centrifuged at 14,000g for
10 min in the cold. The supernatant (soluble ADDLs) was recovered and
transferred to new tubes, and immediately frozen in dry ice and stored at 80 °C
in aliquots of small volume. Several independent ADDLs preparations were assayed
by DB and ELISA comprising ADDLs previously prepared and characterized for
toxicity and synaptic binding13. As Ab monomers, we used the nonaggregated
Ab1-42 diluted in ice-cold MilliQ water (to a final concentration of 100 mM Ab)
and immediately frozen at 80 °C. Recombinant human a-Synuclein (Abcam),
70 mM in 100 mM NaCl, 20 mM Tris-HCl, 1 mM MgCl2 (pH 7.5) was sonicated for
10 min in water bath sonicator, aliquoted and immediately frozen at 80 °C or
oligomerized by incubating the protein solution at 4 °C for 3 or 7 days without
shaking (adapting conditions described64). Purified lysozyme (AppliChem), from
chicken egg white, was dissolved in 25 mM KH2PO4 pH 2 at 45 °C, consecutively
filtered through 0.22 and 0.1 mm pore size syringe filters and assembled as
oligomeric fibrils (0.5 mg ml 1 in 175 mM NaCl/25 mM KH2PO4 pH 2) for 3 h at
50 °C (ref. 65); following a different procedure, lysozyme was also dissolved in
phosphate buffer (PB) 0.1 M at pH 7.4, filtered through 0.1 mm pore size syringe
filters and assembled as oligomers (7 mg ml 1 in PB 0.1 M at pH 7.4) for 4 h at
50 °C (ref. 66). The presence of oligomers in the assembled samples was confirmed
by ELISA, using as primary antibodies the generic anti-oligomer A11 (ref. 21) or
the AbO-specific scFvA13 (ref. 13) (see also Supplementary Information).
Ab immunoproteomic SELDI-TOF MS analysis. The anti-Ab mAbs
(6E10 þ 4G8, 0.125 mg ml 1) (Covance), used as Ab capturing antibodies, were
incubated in a humid chamber for 2 h at room temperature (RT) to allow covalent
binding to the PS20 ProteinChip Array (Bio-Rad Laboratories)27. Unreacted sites
were blocked with Tris-HCl 0.5 M, pH 8 in a humid chamber at RT for 30 min.
Each spot was washed first three times with PBS containing 0.5% (v/v) Triton
X-100 and then twice with PBS. The spots were coated with 5 ml of sample (CM,
straight or 10 concentrated in a Savant Speed Vac) and incubated in a humid
chamber overnight. Each spot was washed first three times with PBS containing
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0.1% (v/v) Triton X-100, twice with PBS and finally with deionized water.
a-cyano-4-hydroxy cinnamic acid (Bio-Rad) was added 1 ml per spot. Mass
identification was made using the ProteinChip SELDI System, Enterprise Edition
(Bio-Rad).
In comparative studies (7WD4 CM versus 7PA2 CM and 7PA2 CM versus
7PA2-A13K CM), the CM was prepared from cells at the same density of plating;
CM was also loaded in polyacrylamide gels, blotted and normalized by Ponceau S
staining of nitrocellulose membrane (detecting the total proteins) and analysed by
WB as described.
Subcellular fractionation. Subcellular fractionation on discontinuous iodixanol
gradients were made as described67. In brief, cells grown to confluence were
pelleted and resuspended in homogenization buffer (10 mM HEPES, pH 7.4, 1 mM
EDTA and 0.25 M sucrose) supplemented with protease inhibitor cocktail (Roche).
Cells were disrupted with a Dounce homogenizer (10 strokes) followed by five
passages through a 27-gauge needle. Nuclei and unbroken cells were pelleted and
the post-nuclear supernatant centrifuged for 1 h at 65,000g. The resultant vesicle
pellets, nominally total microsomes, were resuspended in 0.8 ml of homogenization
buffer. A discontinuous gradient of iodixanol (OptiPrep, Axis-Shield) in 0.25 M
sucrose homogenization buffer, was prepared by underlayering with a syringe
different density solutions ranging from 2.5 to 30%. The resuspended vesicle
fractions were loaded on top of the gradient and centrifuged in a SW40Ti rotor at
40,000g for 2.5 h (4 °C). The resulting gradient was collected in 12 fractions of 1 ml.
Then, each fraction was denaturated and tested by WB for subcellular markers.
Intrabody-AbOs co-IP from subcellular fractions. Freshly prepared subcellular
fractions (or total microsome preparations) were incubated with a cross-linker
solution of dithiobis(succinimidyl propionate) (DSP, Pierce), to a final concentration of 2 mM for 30 min at RT, after that Stop Solution (1 M Tris pH 7.5) was
added to a final concentration of 20 mM for 15 min. Cross-linked subcellular
fractions (or total microsomes) samples were incubated with anti-V5 agarose
affinity gel (Sigma) for 16 h at 4 °C for the immunoprecipitation procedure. After
ice-cold PBS washes (6 times 5,000 g, 5 min each), the immunoprecipitated proteins
were eluted with 4 Tricine sample buffer (Bio-Rad) containing b-mercaptoethanol by 10 min boiling, electrophoresed on Tris-Tricine gel 16.5% (precasted
gels, Bio-Rad). The semidry transfer blot was performed in nitrocellulose membrane filter 0.22 mm and the filter boiled in PBS and subjected to WB analyses
(mAb 6E10 anti-Ab/APP and mAb anti-V5-HRP). Co-IP of small AbOs and
intrabody was analysed in Tris-Tricine gels 16.5% for a better resolution of low
MWs (o50 kDa) WB bands. The full-length APP (B110 kDa) was analysed in
Tris-glycine HCl gels 10% or in Criterion Bis-Tris XT Gels 4–12% (Bio-Rad) upon
elution and boiling of immunoprecipitated proteins with the appropriate sample
buffers (respectively 4 Laemmli Sample buffer or 4 XT sample buffer Bio-Rad
with b-mercaptoethanol).
In vitro assembly interference with synthetic Ab1-42. The human Ab1-42
peptide, HFIP-pretreated (Anaspec) was diluted (5 mM) in anhydrous DMSO and
then in phenol red-free Ham’s F12 medium (PanBiotech) at 100 mM as previously
described68. Aliquots were immediately frozen and stored at 80 °C. Before
aggregation, 100 mM Ab1-42 in Ham’s F12 was diluted at 65 mM by adding
scFvA13 or NaPB buffer (NaCl 100 mM, sodium phosphate buffer pH7, 20 Mm) as
control. The scFvA13 was used at stoichiometric (65 mM) or substoichiometric
(13 mM) ratio with Ab1-42. The aggregation was performed at 22 °C for 24 h. For
disassembly assays, stoichiometric (65 mM) or substoichiometric (13 mM)
concentrations of the scFvA13 or the NaPB buffer as control were added to Ab1-42
in Ham’s F12 (preassembled at the concentration of 100 mM, 22 °C for 24 h)
obtaining a final concentration of 65 mM Ab. The disassembly assay was performed
at 22 °C for 24 h.
Synthetic Ab samples, not boiled nor treated with reducing agents, were loaded
(30 ng per well) in Bis-Tris XT Criterion Gels (Bio-Rad), run in MES buffer and
analysed by WB, as described above by 6E10 (Covance), 4G8 (Covance) or by a
rabbit mAb anti-Ab (D54D2, Cell Signalling).
ER-localized c-secretase activity. Freshly prepared ER-enriched fractions
(from subcellular fractionations) were incubated at 4 °C for determination of basal
APP–CTFs and AICD levels, or incubated at 37 °C for 2 h for APP–CTFs
g-secretase cleavage and de novo AICD generation as previously described31.
Individual fractions were subjected to WB. The detection of AICD was favored by
boiling the membrane filter in PBS.
BFA treatment. Cells (at a confluence of 65–70%) were treated with BFA (10 mM)
for 1 or 4 h. After treatment, cells were washed twice with PBS and lysed or were
left to recover in fresh growth medium for 2 h before lysis. The lysates were
subjected to WB with anti-APP C-terminal antibody (Sigma).
Other treatments. The g-secretase inhibitor treatment of 7PA2 cells were performed with 1 mM of L-685,458 (ref. 29) (purchased from Calbiochem–Millipore)
for 18 h.
The proteasome inhibitor MG132 (Sigma) or the inhibitor of protein synthesis
cycloheximide (Sigma) was administered at 20 and 40 mM, respectively for 6 h.
Hippocampal neuronal cultures. Primary rat hippocampal neurons were prepared from embryonic day 18 pups and maintained in vitro in Neurobasal medium
with B-27 supplement and 0.5 mM L-glutamine for 14 days (14 DIV). Half the
medium was exchanged every 4 days. Neurons (14 DIV) were treated for 16 h with
Neurobasal medium conditioned on confluent dishes of 7PA2 cells or 7PA2-A13K
cells. Cultures were lysed and subjected to WB, as described26.
Immunofluorescence analysis. Cells were plated on glass coverslips and cultured
until reaching B70% confluence, then fixed with 1.9% (v/v) formaldehyde in DPBS
(with Ca2 þ and Mg2 þ ) for 5 min, followed by a postfix for 10 min in 3.7%
formaldehyde at RT. The coverslips were washed, permeabilized with 0.1% Triton
in PBS for 5 min and blocked with 5% FBS in PBS for 1 h at RT. After blocking,
cells were incubated with primary antibodies overnight at 4 °C, and then with
appropriate secondary antibodies (anti-rabbit or anti-mouse Alexa-Fluor 488 or
Alexa-Fluor 594 (Molecular Probes)) for 1 h at RT. Coverslips were mounted with
Aqua-Poly/Mount Reagent (Polysciences) and analysed by confocal microscopy.
Confocal microscopy. Microscopic images were collected at RT using a confocal
laser scanning microscope (TCS SP5, Leica Microsystems) equipped with four laser
lines and with HCX PL APO lambda blue 63.0 1.40 OIL ultraviolet objective.
To exclude cross-talk between the emission spectra of fluorophores, images were
acquired using the sequential acquisition modality. The acquired images were
converted into TIFF files of separated and merged channels by using Leica
Application Suite 6000 (LAS AF Lite).
Statistical analysis. Student’s t-test was used to evaluate differences between two
study groups in normally distributed data (two samples, two tails, equal variance).
Densitometric analysis. WB bands or DB dots images were analysed by using
ScionImage software (NIH Image for Windows, Beta 4.0.2).
Western Blot analysis. Full-lenght images of western blot are shown in
Supplementary Figs 14,15.
Preparation of recombinant scFvA13 and scFvIm3. The cDNA of anti-AbOs
scFvA13 and scFvIm3 (ref. 13) were cloned by restriction with the enzymes
BssHII/NheI into the pETM13 vector (modified in the polylinker), and
transformed in BL21(DE3)pLysS E. coli (Novagen).
The recombinant scFv proteins were prepared following established procedures
for the expression and refolding of another SPLINT-derived scFv in the cytoplasm
of E. coli 69.
The purification of the refolded scFvs were achieved through a cation exchange
chromatography, performed on HiTrap SP column (5 ml, GE), equilibrated with
20 mM sodium phosphate buffer, pH 7 (for scFvA13) and pH 6.5 (for scFvIm3) as
A buffer. The elution of the sample was achieved with a linear gradient from 0 to
70% of B buffer (A buffer þ 1 M NaCl).
The sample was then dialysed against PBS and purified on a size exclusion
chromatography column, a Superdex 75 (GE), equilibrated with PBS.
Antibodies used in WB, DB, IP and immunofluorescence analyses. Anti-Ab/
APP 4G8 (mouse mAb, Covance), anti-Ab/APP 6E10 (mouse mAb, Covance),
anti-AbOs scFvA13 and scFvIm3 (ref. 13), anti-oligomer A11 (ref. 21) (rabbit pAb
Millipore), anti-Ab (rabbit mAb, Cell Signaling), anti a-Synuclein (mouse mAb,
Abcam), anti-APP C-terminal fragment (rabbit pAb, Sigma), anti-APP N-terminal
22C11 (mouse mAb, Millipore), anti-V5 (mouse mAb Invitrogen, and rabbit pAb
Sigma), anti-V5-HRP (mouse mAb, Sigma), anti-His tag (mouse mAb Millipore),
anti-KDEL (mouse mAb, Assay Design/Stressgen) (kind gift of Prof. R. Sitia, HSR
San Raffaele, Milan), anti-mTOR (rabbit pAb, Cell Signaling), anti-phosphomTOR (rabbit pAb, Cell Signaling), anti-P70S6K (rabbit pAb, Cell Signaling) (kind
gift of Dr G. Amadoro, CNR, Rome), anti-phospho-P70S6K (rabbit pAb, Cell
Signaling), anti ERK1/2 (rabbit pAb, Cell Signaling), anti phosho-ERK1/2 (rabbit
pAb, Cell Signaling), anti phospho-CREB (rabbit pAb, Cell Signaling), anti b-Actin
(mouse mAb and rabbit pAb, Sigma), anti-calnexin (rabbit pAb, Sigma), antiGM130 (mouse mAb, Covance), anti-Golgi 58K (mouse mAb, Sigma), anti-Rab3A
(mouse mAb, Sigma), anti-Rab5 (rabbit pAb, Abcam), anti-Rab7 (goat pAb, Santa
Cruz), anti-TGN46 (rabbit, pAb), anti-LAMP-1 (rabbit pAb, Abcam). Anti-Rab5,
anti-Rab7, anti-TGN46 are kind gift of Dr V. Triaca, CNR, Rome.
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Acknowledgements
We thank Dr D.J. Selkoe (Harvard Medical School, Boston) for providing 7PA2 and
7WD4 cells, Dr C.A. Lemere (Harvard Medical School, Boston) for helpful discussions,
Maria Teresa Ciotti (IFT-CNR, Rome) for hippocampal neuron cultures, Francesca
Malerba, Francesca Paoletti and Bruno Bruni, (EBRI, Rome) for advice and help in
preparation of recombinant purified scFvA13, Emanuela Colla (SNS, Pisa) for advice on
a-Synuclein assays. This work was supported by grants from Alzheimer’s Association
(IIRG-06-27105), FIRB MIUR (RBAP10L8TY), Fondazione Roma, Human Brain Project
(HBP) Neuroantibodies to A.C. and from Alzheimer’s Association (NIRG-12-237751)
to G.M.
Author contributions
G.M. and A.C. designed the study, analysed data and wrote the manuscript; G.M.,
A.L., A.M. and N.K. performed the experiments and analysed data; V.A. performed
immunoproteomic measures; L.B. and R.G. analysed immunoproteomic data.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/nature
communications
Competing financial interests: The authors declare no competing financial interests.
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How to cite this article: Meli, G. et al. Conformational targeting of intracellular
Ab oligomers demonstrates their pathological oligomerization inside the endoplasmic
reticulum. Nat. Commun. 5:3867 doi: 10.1038/ncomms4867 (2014).
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